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Substrate Specificity of Mutants of the Hydroxynitrile Lyase from Manihot esculenta
Author(s) -
Bühler Holger,
Effenberger Franz,
Förster Siegfried,
Roos Jürgen,
Wajant Harald
Publication year - 2003
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200390033
Subject(s) - chemistry , diisopropyl ether , cyanohydrin , active site , ether , substrate (aquarium) , yield (engineering) , lyase , stereochemistry , organic chemistry , enzyme , catalysis , oceanography , materials science , metallurgy , geology
Several tryptophan128‐substituted mutants of the hydroxynitrile lyase from Manihot esculenta (MeHNL) are constructed and applied in the MeHNL‐catalyzed addition of HCN to various aromatic and aliphatic aldehydes as well as to methyl and ethyl ketones to yield the corresponding cyanohydrins. The mutants (especially MeHNL‐W128A) are in most cases superior to the wild‐type (wt) enzyme when diisopropyl ether is used as the solvent. Substitution of tryptophan128 by an alanine residue enlarges the entrance channel to the active site of MeHNL and thus facilitates access of sterically demanding substrates to the active site, as clearly demonstrated for aromatic aldehydes, especially 3‐phenoxybenzaldehyde. These experimental results are in accordance with the X‐ray crystal structure of MeHNL‐W128A. Aliphatic aldehydes, surprisingly, do not demonstrate this reactivity dependence of mutants on substrate bulkiness. Comparative reactions of 3‐phenoxybenzaldehyde with wtMeHNL and MeHNL‐W128A in both aqueous citrate buffer and a two‐phase system of water/methyl tert ‐butyl ether again reveal the superiority of the mutant enzyme: 3‐phenoxybenzaldehyde was converted quantitatively into a cyanohydrin nearly independently of the amount of enzyme present, with a space‐time yield of 57 g L −1 h −1 .